Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2000 Jun;11(6):1989-2005.
doi: 10.1091/mbc.11.6.1989.

Identification of a novel family of nonclassic yeast phosphatidylinositol transfer proteins whose function modulates phospholipase D activity and Sec14p-independent cell growth

X Li  1 , S M Routt, Z Xie, X Cui, M Fang, M A Kearns, M Bard, D R Kirsch, V A Bankaitis
Affiliations
Free PMC article

Identification of a novel family of nonclassic yeast phosphatidylinositol transfer proteins whose function modulates phospholipase D activity and Sec14p-independent cell growth

X Li et al. Mol Biol Cell. 2000 Jun.
Free PMC article

Abstract

Yeast phosphatidylinositol transfer protein (Sec14p) is essential for Golgi function and cell viability. We now report a characterization of five yeast SFH (Sec Fourteen Homologue) proteins that share 24-65% primary sequence identity with Sec14p. We show that Sfh1p, which shares 64% primary sequence identity with Sec14p, is nonfunctional as a Sec14p in vivo or in vitro. Yet, SFH proteins sharing low primary sequence similarity with Sec14p (i.e., Sfh2p, Sfh3p, Sfh4p, and Sfh5p) represent novel phosphatidylinositol transfer proteins (PITPs) that exhibit phosphatidylinositol- but not phosphatidylcholine-transfer activity in vitro. Moreover, increased expression of Sfh2p, Sfh4p, or Sfh5p rescues sec14-associated growth and secretory defects in a phospholipase D (PLD)-sensitive manner. Several independent lines of evidence further demonstrate that SFH PITPs are collectively required for efficient activation of PLD in vegetative cells. These include a collective requirement for SFH proteins in Sec14p-independent cell growth and in optimal activation of PLD in Sec14p-deficient cells. Consistent with these findings, Sfh2p colocalizes with PLD in endosomal compartments. The data indicate that SFH gene products cooperate with "bypass-Sec14p" mutations and PLD in a complex interaction through which yeast can adapt to loss of the essential function of Sec14p. These findings expand the physiological repertoire of PITP function in yeast and provide the first in vivo demonstration of a role for specific PITPs in stimulating activation of PLD.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Sec14p pathway in yeast. The PtdCho- and PtdIns-bound forms of Sec14p are proposed to independently regulate inositol and choline phospholipid metabolism (reviewed by Kearns et al., 1998a). PtdCho-bound Sec14p down-regulates flux through the diacylglycerol (DAG)-utilizing CDP-choline pathway for PtdCho biosynthesis. PtdIns-bound Sec14p stimulates PtdIns-4-P synthesis via the Pik1p pathway. PtdIns-4-P is a precursor for PtdIns-4,5-P2, which is required for activation of PLD. PLD hydrolyzes PtdCho to PtdOH and choline. PtdOH is dephosphorylated by PtdOH-phosphohydrolases to DAG. Evidence to support individual roles for PtdCho, DAG, PtdOH, PtdIns-4-P, and PtdIns-4,5-P2 in modulating yeast Golgi secretory function exists. However, the lipid regulators that directly interact with the Sec14p pathway are unclear. Bypass-Sec14p mutations used in this study inactivate choline kinase (CKIase; cki1), choline phosphate cytidylyltransferase (CCTase; pct1), the SacIp phosphoinositidase (sac1), and the Kes1p oxysterol-binding protein homologue (kes1). Known execution points for these enzymes/proteins are given. The precise execution point for Kes1p remains unclear.
Figure 2
Figure 2
SFH proteins can rescue growth and secretory defects associated with Sec14p dysfunction. (A) Schematic alignment of SFH gene products (also identified by their yeast genome database accession numbers) with Sec14p. Regions of homology are shaded, and the primary sequence identities/similarities shared by individual SFH gene products with Sec14p are given as percentages. (B) Expression of SFH2, SFH4, and SFH5 genes rescues the growth defects of sec14ts and Δsec14 yeast mutants. Growth properties of a sec14-1ts yeast strain (CTY1-1A) carrying YEp(SEC14) and YEp(URA3) as respective positive and negative controls for phenotypic complementation of the sec14-1ts growth defect at 37°C are as indicated. Growth properties of strain CTY1-1A carrying YEp(SFH2), YEp(SFH3), YEp(SFH4), or YEp(SFH5) are also shown. All yeast strains were streaked for isolation onto YPD plates and incubated at either a permissive (25°C) or a restrictive (37°C) temperature for sec14-1ts strains, as indicated. After 48 h of incubation, the growth results were recorded. YEp(SFH2) and YEp(SFH4) were nearly as effective as YEp(SEC14) in rescuing growth of the sec14-1ts strain at 37°C. YEp(SFH5) was less effective, and YEp(SFH3) was ineffective. (C) Expression of SFH2, SFH3, SFH4, and SFH5 increases invertase secretion efficiency in sec14ts strains at 37°C. Invertase secretion indices (i.e., measures of secretory function) were determined (see MATERIALS AND METHODS). Wild-type strains secrete invertase efficiently (secretion index ≥ 0.90), and Sec14p-insufficient cells secrete invertase poorly (secretion index ≤ 0.20). Each YEp(SFH) expression cassette markedly improved secretory pathway function under conditions of Sec14p deficiency. Values represent averages of at least three independent trials with triplicate determinations for each strain.
Figure 2
Figure 2
SFH proteins can rescue growth and secretory defects associated with Sec14p dysfunction. (A) Schematic alignment of SFH gene products (also identified by their yeast genome database accession numbers) with Sec14p. Regions of homology are shaded, and the primary sequence identities/similarities shared by individual SFH gene products with Sec14p are given as percentages. (B) Expression of SFH2, SFH4, and SFH5 genes rescues the growth defects of sec14ts and Δsec14 yeast mutants. Growth properties of a sec14-1ts yeast strain (CTY1-1A) carrying YEp(SEC14) and YEp(URA3) as respective positive and negative controls for phenotypic complementation of the sec14-1ts growth defect at 37°C are as indicated. Growth properties of strain CTY1-1A carrying YEp(SFH2), YEp(SFH3), YEp(SFH4), or YEp(SFH5) are also shown. All yeast strains were streaked for isolation onto YPD plates and incubated at either a permissive (25°C) or a restrictive (37°C) temperature for sec14-1ts strains, as indicated. After 48 h of incubation, the growth results were recorded. YEp(SFH2) and YEp(SFH4) were nearly as effective as YEp(SEC14) in rescuing growth of the sec14-1ts strain at 37°C. YEp(SFH5) was less effective, and YEp(SFH3) was ineffective. (C) Expression of SFH2, SFH3, SFH4, and SFH5 increases invertase secretion efficiency in sec14ts strains at 37°C. Invertase secretion indices (i.e., measures of secretory function) were determined (see MATERIALS AND METHODS). Wild-type strains secrete invertase efficiently (secretion index ≥ 0.90), and Sec14p-insufficient cells secrete invertase poorly (secretion index ≤ 0.20). Each YEp(SFH) expression cassette markedly improved secretory pathway function under conditions of Sec14p deficiency. Values represent averages of at least three independent trials with triplicate determinations for each strain.
Figure 2
Figure 2
SFH proteins can rescue growth and secretory defects associated with Sec14p dysfunction. (A) Schematic alignment of SFH gene products (also identified by their yeast genome database accession numbers) with Sec14p. Regions of homology are shaded, and the primary sequence identities/similarities shared by individual SFH gene products with Sec14p are given as percentages. (B) Expression of SFH2, SFH4, and SFH5 genes rescues the growth defects of sec14ts and Δsec14 yeast mutants. Growth properties of a sec14-1ts yeast strain (CTY1-1A) carrying YEp(SEC14) and YEp(URA3) as respective positive and negative controls for phenotypic complementation of the sec14-1ts growth defect at 37°C are as indicated. Growth properties of strain CTY1-1A carrying YEp(SFH2), YEp(SFH3), YEp(SFH4), or YEp(SFH5) are also shown. All yeast strains were streaked for isolation onto YPD plates and incubated at either a permissive (25°C) or a restrictive (37°C) temperature for sec14-1ts strains, as indicated. After 48 h of incubation, the growth results were recorded. YEp(SFH2) and YEp(SFH4) were nearly as effective as YEp(SEC14) in rescuing growth of the sec14-1ts strain at 37°C. YEp(SFH5) was less effective, and YEp(SFH3) was ineffective. (C) Expression of SFH2, SFH3, SFH4, and SFH5 increases invertase secretion efficiency in sec14ts strains at 37°C. Invertase secretion indices (i.e., measures of secretory function) were determined (see MATERIALS AND METHODS). Wild-type strains secrete invertase efficiently (secretion index ≥ 0.90), and Sec14p-insufficient cells secrete invertase poorly (secretion index ≤ 0.20). Each YEp(SFH) expression cassette markedly improved secretory pathway function under conditions of Sec14p deficiency. Values represent averages of at least three independent trials with triplicate determinations for each strain.
Figure 3
Figure 3
SFH proteins are atypical PITPs. The ability of each SFH protein to transfer [3H]PtdIns (A and C) or [14C]PtdCho (B and D) was determined in either cytosol fractions prepared from salt-stripped yeast membranes (A and B) or cytosol prepared from E. coli strains expressing recombinant SFH gene product (C and D). Activity is represented as the percentage of total input [3H]PtdIns or [14C]PtdCho transferred from donor membranes to unlabeled acceptor membranes during the course of the experiment (see MATERIALS AND METHODS). Cytosol values are presented as amounts of protein added to the assay cocktail. For the yeast cytosol experiments, the proteins of interest were expressed in strain CTY303 (Table 1), which has no detectable endogenous PtdIns- or PtdCho-transfer activity. CTY303/YEp(URA3) cytosol was prepared and used as a negative control. Assay blanks represent addition of buffer alone to the transfer assay reactions. ♦, Sec14p; ▴, Sfh2p; ▪, Sfh3p; ●, Sfh4p; ×, Sfh5p; ○, vector control. In experiments that used yeast cytosol, 14,155–17,319 cpm of input [3H]PtdIns and 14,963–22,035 cpm of input [14C]PtdCho was used in each assay. Background values for these PtdIns- and PtdCho-transfer assays were in the range of 521–697 and 76–159 cpm, respectively. All samples contained <170 mM KCl, a salt concentration that itself had no effect on background or signal in these assays. In experiments that used E. coli cytosol, 15,034–15,411 cpm of input [3H]PtdIns and 18,950–22,531 cpm of input [14C]PtdCho was used in each assay. Background values for assays that used recombinant SFH proteins were in the range of 501–601 for the PtdIns-transfer assays and 547–578 for the PtdCho-transfer assays. No KCl was present in any assays that used E. coli cytosol. These data are representative of at least five independent experiments.
Figure 4
Figure 4
Phenotypic consequences of SFH gene product insufficiency. (A) The sec14-1ts strain CTY1-1A (SFH), CTY1-1A derivatives carrying individual disruptions of each SFH gene as indicated at left, and a sec14-1ts strain collectively disrupted for SFH2, SFH3, SFH4, and SFH5 (Δsfh) were streaked for isolation on YPD agar and incubated at the indicated temperatures. Growth was scored after 48 and 72 h of incubation. Growth of SEC14 yeast was not affected by individual or aggregate disruption of SFH genes at any of the temperatures tested (our unpublished results). Disruption of SFH2 or SFH3 reduced the restrictive temperature of sec14-1ts strains, and en bloc deletion of the SFH genes exerted a more pronounced effect. Growth phenotypes of a sec14-1ts, Δspo14 mutant are presented for comparison. (B) Yeast strains (the sec14 and bypass-Sec14p genotypes indicated at top) carrying the designated individual sfh gene disruptions, or en bloc disruptions of SFH2, SFH3, SFH4, and SFH5 (Δsfh) (at left), were streaked for isolation on YPD agar and incubated at 37°C for 48 h. Growth phenotypes of Δspo14 derivatives of these bypass-Sec14p mutants are also presented for comparison. The Δspo14 and Δsfh genetic backgrounds were nonpermissive for bypass Sec14p.
Figure 5
Figure 5
SFH gene products contribute to PLD activity in vivo. (A) PLD requirement for YEp(SFH)-mediated rescue of sec14-associated growth defects. The sec14-1ts, Δspo14 strain CTY1079 was transformed with the indicated YEp(SFH) plasmids and YEp(SEC14) and YEp(URA3) plasmids as positive and negative controls, respectively. The corresponding transformants were streaked for isolated colonies on YPD agar and incubated at temperatures permissive (26°C) or restrictive (37°C) for the sec14-1ts, Δspo14 strain, as indicated. Results were scored after 48 h of incubation. YEp(SFH2) elicited a clear phenotypic rescue of sec14-1ts growth defects, whereas YEp(SFH3), YEp(SFH4), and YEp(SFH5) were unable to effect any relief of the sec14-associated growth defects in the absence of functional PLD activity. (B) PLD activation in Δsfh yeast strains as measured by choline excretion. Equal cell numbers of the sec14-1ts, cki1 strain CTY160 and its congenic sec14-1ts, cki1, Δsfh derivative CTY1427 were spotted on a lawn of the choline auxotrophic strain CTY1289 plated onto choline-free minimal agar. The plate was incubated at 30°C for 48 h. An inoculum of the isogenic Δspo14 derivative strain CTY1099 was also spotted onto the indicator lawn, and the absence of choline cross-feeding of the indicator establishes that the choline excreted by the CTY160 and CTY1289 cells was generated by the action of PLD. (C) Chemical quantification of choline excretion. The appropriate yeast strains were grown to midlogarithmic growth phase in minimal defined medium supplemented with inositol (100 μM) but lacking choline at 26°C. The cells were harvested, washed, and resuspended in fresh medium and incubated at 33.5°C for 3 h. A choline oxidase–coupled chemical assay was used to measure the choline concentration of culture supernatant (see MATERIALS AND METHODS). Relevant genotypes of the strains are given below the bars. Data represent triplicate determinations from at least three independent experiments. (D) PLD activation in Δsfh yeast strains as measured by PtdOH production. The appropriate yeast strains were radiolabeled to steady state with [32P]orthophosphate (10 μCi/ml) at 26°C in inositol- and choline-replete minimal medium (Xie et al., 1998). Cells were subsequently washed and incubated in radiolabel-free medium for 3 h at 33.5°C, and phospholipids were extracted and resolved (Xie et al., 1998). PtdOH was identified and quantified by phosphorimaging. Values are expressed as [32P (cpm) incorporated into PtdOH divided by total 32P (cpm) incorporated into extractable phospholipid] times 100%. The data were normalized by measuring relative rates of phosphate incorporation into phospholipid as a function of cell number for each strain. Under these experimental conditions, the following values of 32P (cpm) incorporation into extractable phospholipid per OD600 cells were obtained: CTY182 (SEC14, SFH), 8500 − 245; CTY1-1A (sec14-1ts, SFH), 17,000 − 1470; CTY1079 (sec14-1ts, Δspo14), 24,000 − 2400; CTY1118 (sec14-1ts, Δsfh), 15,000 − 645. These data represent the averages of at least three independent experiments.
Figure 6
Figure 6
SFH gene products and PLD activity in broken cell preparations. The sec14-1ts, cki1 strain CTY160, its isogenic Δspo14 partner CTY1099, and its congenic Δsfh derivative CTY1427 were used in these experiments. These strains were cultured in YPD at 26°C until they reached early logarithmic growth phase. The cultures were then shifted to 33.5°C for 3 h, cells were harvested and broken by glass-bead lysis, and clarified lysates were prepared. PLD activity was assayed by monitoring conversion of input NBD-PtdCho substrate to NBD-PtdOH, as described by Waksman et al. (1996) (see MATERIALS AND METHODS). (A) TLC of PLD assay products. The products of PLD activity assays were resolved by one-dimensional TLC and visualized by illumination under UV light. The positions of input NBD-PtdCho substrate and NBD-PtdOH product are indicated at right. Identification of each species was made by comparison with the chromatographic properties of NBD-PtdCho and NBD-PtdOH standards. Relevant genotypes of yeast strains that served as sources of lysate for each corresponding assay are given above each lane. The sec14-1ts control lysate clearly supported conversion of NBD-PtdCho to NBD-PtdOH that was PLD dependent, as judged by the inability of Δspo14 lysate to support this reaction. Relative to the sec14-1ts, SFH control, lysate from the Δsfh strain effected only a weak, but detectable, conversion of NBD-PtdCho to NBD-PtdOH. (B) Quantification of in vitro PLD activity. Fluorometry was used to quantify NBD fluorescence in PtdCho and PtdOH species resolved from PLD activity assays by TLC. Relevant genotypes of the yeast strains from which the test lysates were prepared are given below each bar. The results are expressed as (NBD fluorescence recovered in PtdOH divided by NBD fluorescence recovered in PtdOH and PtdCho) times 100%. All fluorescence values obtained fell in the linear range of fluorometric detection, as assessed by fluorescence counting of serial dilutions of known quantities of NBD phospholipid standards. Measured values are given above each corresponding data bar (n ≥ 3).
Figure 7
Figure 7
Sfh2p and PLD partially localize to endosomes. Yeast cells expressing Sfh2p-GFP or PLD-GFP were stained with FM 4-64 under conditions in which the styryl dye is localized to endosomes (see MATERIALS AND METHODS). Separate images of GFP and FM 4-64 fluorescence were captured from the same sets of living yeast cells, and arrowheads indicate some examples of FM 4-64 structures (B and F) decorated with either Sfh2p-GFP (A) or PLD-GFP (E). Computer merge of these images confirms the colocalization of Sfh2p-GFP and GFP-PLD with FM 4-64 structures (our unpublished results). Because the Sfh2p-GFP fluorescence is rather dim, only the cells in the precise focal plane are apparent in A, whereas other cells in the approximate focal plane are also visible when the more intense FM 4-64 fluorescence is monitored in B. C, D, G, and H depict fluorescence bleed controls. C and G show the fluorescence pattern when the fields depicted in A and E, respectively, were excited for GFP fluorescence and viewed through the filter used to record FM 4-64 fluorescence. Reciprocally, D and H show the fluorescence pattern when the fields depicted in B and F, respectively, were excited for FM 4-64 fluorescence and viewed through the filter used to record GFP fluorescence. No fluorescence bleed was detected between the GFP and FM 4-64 channels.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Aitken JF, van Heusden GPH, Temkin M, Dowhan W. The gene encoding the phosphatidylinositol transfer protein is essential for cell growth. J Biol Chem. 1990;265:4711–4717. - PubMed
    1. Atkinson KD. Saccharomyces cerevisiae recessive suppressor that circumvents phosphatidylserine deficiency. Genetics. 1984;108:533–543. - PMC - PubMed
    1. Bankaitis VA, Aitken JR, Cleves AE, Dowhan W. An essential role for a phospholipid transfer protein in yeast Golgi function. Nature. 1990;347:561–562. - PubMed
    1. Bankaitis VA, Fry MR, Cartee RT, Kagiwada S. Molecular Biology Intelligence Unit. Austin, TX: RG Landes; 1996. Phospholipid transfer proteins: emerging roles in vesicle trafficking, signal transduction, and metabolic regulation; pp. 94–97.
    1. Bankaitis VA, Malehorn DE, Emr SD, Greene R. The Saccharomyces cerevisiae SEC14 gene encodes a cytosolic factor that is required for transport of secretory proteins from the yeast Golgi complex. J Cell Biol. 1989;108:1271–1281. - PMC - PubMed

Publication types

MeSH terms